Genetic engineering has allowed both small and large peptides and proteins to be expressed in a number of different systems. Whilst some proteins manufactured in this way are suitable for therapeutic use, small differences in the way that the proteins are modified by the cells after they are made mean that often the intended therapeutic proteins are not exactly the same as those native proteins found in the animal or human to be treated.
Different systems produce different glycosylation patterns. For example some yeast expression systems do not glycosylate at all, and frog oocytes usually glycosylate in a different way to Chinese hamster ovaries. As a result, when these systems are used it is often desirable to subsequently modify the glycosylation pattern of the peptide or protein.
Much attention has focussed on enzymatic modification of proteins rather than chemical modification as enzymes tend to be able to carry out specific reactions in relatively mild conditions in which the protein to be modified will generally be stable. Chemical modification usually requires relatively harsher conditions and may, in some instances, be non-specific, and therefore give a number of different products that may need to be separated by, for example, chromatography.
Although chemical methods tend to be harsher and less specific than enzymatic methods, chemical modification can be carried out relatively quickly, with a minimum of laboratory equipment and with minimal expense. Methods are available for chemically modifying proteins, for example to attach sugars, and these methods generally involve chemical reaction of a side chain functional group such as asparagines, serine or threonine. Other amino acid side chain functionalities may also be reacted but further methods are desired and there is a need in the art for chemical methods of protein modification that are specific and which do not irreversibly affect amino acid side chains other than the target of modification.
It would also be useful if these chemical methods utilised a reaction that proceeded rapidly while preserving all other amino acid side chains.
The Merrifield solid state peptide synthesis has allowed the chemical synthesis of polypeptides to be carried out more efficiently than traditional solution chemistry. However, although the reactions are very nearly quantitative, small incremental losses of product and deviations from the desired amino acid order result in a practical limitation that render the method unfeasible for synthesis of polypeptides greater than about 70 residues in length. Thus, a large protein is generally chemically synthesised by coupling two or more synthesisable segments of the sequence together by native chemical ligation, a technique to react a peptide containing a C-terminal thioester with another peptide containing an N-terminal cysteine, in the presence of an exogenous thiol catalyst. Whilst this reaction proceeds with near quantitative yields, it is limited by the requirement that the resulting peptide had a cysteine residue at an appropriate position in the sequence.
It is an object of the invention to provide a method for chemically modifying proteins and peptides which addresses any limitations, needs or problems highlighted herein with the prior art methods or at least to provide the public and research community with a useful choice.
Documents cited in this specification are hereby incorporated by reference although no admission is made that any constitute prior art. The discussion of the documents states what their authors have asserted, and the applicants reserve the right to challenge the accuracy of the cited documents. Although prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art in any country.
It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning; that it will be taken to mean an inclusion of not only the listed components or steps it directly references, but also other non-specified components or elements. This rationale applies when the related terms ‘comprised’ or ‘comprising’ are used in relation to one or more steps in a method or process.